Chemiluminescence enhancement of enzyme-activated decomposition of enzymatically cleavable chemiluminescent 1,2-dioxetanes
1,2- Dioxetane derivatives
Enhancement of chemiluminescent assays Patent #: 5994073
ApplicationNo. 10924980 filed on 08/25/2004
US Classes:436/546, Fluorescent label436/172, With fluorescence or luminescence436/904, OXIDATION - REDUCTION INDICATORS435/968, HIGH ENERGY SUBSTRATES (E.G., FLUORESCENT, CHEMILUMINESCENT, RADIOACTIVE, ETC.)435/21, Involving phosphatase435/28, Involving peroxidase549/507, One double bond between ring members of the hetero ring549/497, Chalcogen attached indirectly to the hetero ring by nonionic bonding549/504, Halogen attached directly or indirectly to the hetero ring by nonionic bonding435/6, Involving nucleic acid435/7.9Assay in which an enzyme present is a label
ExaminersPrimary: Ceperley, Mary E.
Assistant: Haq, Shafiqul
Attorney, Agent or Firm
Foreign Patent References
International ClassesG01N 33/532
The present invention relates to a method for intensifying chemiluminescence of 1,2-dioxetane and a composition for chemiluminescence. Themethod according to the present invention is effective for a high-sensitive analysis for a biological component, an enzyme, a chemical product or the like, and the composition for chemiluminescence according to the present invention can suitably be usedas a substrate reagent for an enzyme immunoassay.
A water-soluble chemiluminescent material having a 1,2-dioxetane structure is a very useful compound under the condition under which a protic solvent is used such as an analysis of a biological component, and the demand becomes increasingly highin recent years. However, in a case where 1,2-dioxetane is used alone in the protic solvent, only very low luminous efficiency can be obtained, and accordingly, even if it is intended to be used for immunoassay for a clinical examination, nochemiluminescence intensity which can be practically used could be obtained. Therefore, it has been necessary to intensify the chemiluminescence of water-soluble chemiluminescent dioxetane. In the present specification, materials useful to increasechemiluminescence intensity of a chemiluminescent compound are generically called as sensitizers or enhancers regardless of whether or not they are directly concerned with electronic energy transfer.
Heretofore, as the enhancers to 1,2-dioxetane which is decomposed into two carbonyl-containing compounds and discharges electronic energy to emit light, a cationic surfactant (for example JP-B-7-121237), a mixture of a cationic surfactant and afluorescent material (JP-B-7-91536), a vinylbenzyl quaternary ammonium salt polymer (for example JP-B-7-31201) and a polycationic high-polymer phosphonium salt (Japanese Patent No. 2648423) have been proposed. Further, there is an example wherein aquaternary phosphonium salt is added to a chemiluminescence system of acridinium ester (JP-A-5-79985).
However, a 1,2-dioxetane derivative having a furan ring as represented by the formula 1 (for example JP-A-9-216887) to be used in the present invention, is a chemiluminescent material excellent in heat stability, which is not decomposed into twocompounds at the time of luminescence. However, the most suitable sensitizer to increase the chemiluminescence intensity has not been reported up to now.
Under these circumstances, it is an object of the present invention to provide a chemiluminescence method, which is the most suitable for the chemiluminescence of a 1,2-dioxetane derivative having a furan ring. Further, it is an object of thepresent invention to provide a composition for chemiluminescence, which can also be used as a substrate reagent for an enzyme immunoassay, is excellent in heat stability, and makes a high-sensitive analysis possible.
The present inventors have conducted extensive studies on the above problems, and as a result, achieved the present invention. Namely, the present invention provides a chemiluminescence method characterized in that when a 1,2-dioxetanederivative of the formula 1:
##STR00002## wherein each of R1, R2, R3, R4 and R5 which are independent of one another, is a hydrogen atom, an alkyl group or an aryl group, or each pair of R2 and R3, and R4 and R5 which areindependent of each other, may form together a cyclic alkyl group, R6 is a hydroxyl group, an alkoxyl group, an aralkyloxy group, a group represented by --OSi(R8R.sup.9R.sup.10) (provided that each of R8, R9 and R10 which areindependent of one another, is an alkyl group or an aryl group), a phosphate group or a group represented by S(C=O)R11 (provided that R11 is an alkyl group or an aryl group), and R7 is a hydrogen atom, a halogen atom, an alkyl group oran alkoxyl group, is made to generate chemiluminescence by means of an activator selected from the group consisting of a base, an acid, a salt, a fluorine compound, an enzyme, a catalyst and an amine compound, a cationic surfactant and a fluorescentmaterial are made to coexist.
Further, the present invention provides a composition for chemiluminescence comprising a 1,2-dioxetane derivative of the above formula 1, a cationic surfactant and a fluorescent material.
Now, the present invention will be described in further detail.
Firstly, the 1,2-dioxetane derivative of the formula 1 will be described.
The alkyl group as the substituent is a C1-20 straight chain, branched or cyclic alkyl group, and it may, for example, be a straight chain group such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl or eicosanyl, or a group having these alkyl groups appropriately bonded in a branched form or in a cyclic form. Such an alkyl group may further have a substituent. Forexample, it may be substituted by e.g. a hydroxyl group, an alkoxyl group or an aryl group.
As the aryl group, a C6-20 aromatic hydrocarbon group such as a phenyl or naphthyl group, or a heteroaryl group having from 1 to 5 nitrogen atoms, oxygen atoms or sulfur atoms in the ring such as a furyl, thienyl or pyridyl group may, forexample, be mentioned. As the alkoxyl group, one having from 1 to 5 C1-20 alkoxy groups bonded in a straight chain form or in a branched form, such as methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy,ethoxyethoxy, ethoxypropoxy or methoxyethoxyethoxy, may, for example, be mentioned. As the aralkyloxy group, a C7-20 group such as a benzyloxy or phenethyloxy group may, for example, be mentioned. As the halogen atom, fluorine, chlorine or brominemay, for example, be mentioned.
As the 1,2-dioxetane derivative having such substituents of the formula 1, specifically, 5-t-butyl-4,4-dimethyl-l-(3'-phosphoryloxy)phenyl-2,6,7-trioxabicyclo[3.2- .0]heptane disodium salt or phosphoric acidmono-[5-(5-t-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]hept-1-yl)-2-ch- lorophenyl]ester disodium salt may, for example, be mentioned.
The activator to be used in the present invention is used to let the 1,2-dioxetane derivative of the formula 1 generate luminescence. It is not particularly limited so long as it is one which acts on the group R6 in the 1,2-dioxetanederivative of the formula 1 to convert the derivative into a carbonyl compound via an unstable oxide intermediate and makes the derivative generate chemiluminescence at the same time. For example, if R6 is a hydroxyl group, a base may suitably beused, and if R6 is a group represented by --OSi(R8R.sup.9R.sup.10) (provided that each of R8, R9 and R10 which are independent of one another, is an alkyl group or an aryl group), tetrabutylammonium fluoride may suitably be usedas the activator. In a case where the 1,2-dioxetane derivative of the formula 1 is used as a substrate for an enzyme immunoassay, various enzymes such as alkaline phosphatase, arylesterase, acetylcholinesterase, peroxidase and galactosidase may suitablybe used as the activator.
As the cationic surfactant to be used in the present invention, it is not particularly limited so long as it is one having an effect for intensifying chemiluminescence of the 1,2-dioxetane of the formula 1 together with a fluorescent material asmentioned hereinafter. As a suitable cationic surfactant, for example, poly(benzyltributyl)ammonium chloride contained in commercially available Emerald-II™ (manufactured by TROPIX, Inc.) or a quaternary ammonium salt or a quaternary phosphoniumsalt of the formula 2:
##STR00003## wherein one of R12, R13, R14 and R15 is a C8-20 alkyl group or a C8-20 aryl group, and the other three are each independently a C1-8 alkyl group, a benzyl group or a phenyl group, M.sup. represents a positively charged nitrogen atom or phosphorus atom, and X- represents a halogen ion may be mentioned.
In the formula 2, the C8-20 alkyl group is a C8-20 straight chain or branched alkyl group, and it may, for example, be a straight chain group such as octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,octadecyl, nonadecyl or eicosanyl, or a group having these alkyl groups appropriately bonded in a branched form. The C1-8 alkyl group is a C1-8 straight chain or branched alkyl group, and it may, for example, be a straight chain such asmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl, or a group having these alkyl groups appropriately bonded in a branched form. As the C8-20 aryl group, a C8-20 aromatic hydrocarbon group such as a naphthyl group, or a heteroarylgroup having from 1 to 5 nitrogen atoms, oxygen atoms or sulfur atoms in the ring may, for example, be mentioned. As the halogen ion, a fluorine ion, a chlorine ion or a bromine ion may, for example, be mentioned.
As a suitable example of the quaternary ammonium salt of the formula 2 having substituents as mentioned above, cetyl trimethylammonium bromide (CTAB), benzalkonium chloride or benzethonium chloride may be mentioned. Further, as a suitableexample of the quaternary phosphonium salt, tri-n-butylhexadecylphosphonium bromide, ethyltriphenylphosphonium bromide, triheptylphenylphosphonium bromide or tetradecyltriphenylphosphonium bromide may be mentioned. Among them,tri-n-butylhexadecylphosphonium bromide is particularly preferred.
The fluorescent material to be used in the present invention is not particularly limited so long as it is one having an effect for intensifying chemiluminescence of the 1,2-dioxetane derivative of the formula 1 together with the cationicsurfactant. For example, fluorescein, a fluorescein derivative (such as sodium fluorescein, aminofluorescein or fluorescein isothiocyanate (FITC)), a Bodipy dye, Oregon Green 488, Oregon Green 514, Rhodol Green (trademark), Alexa Fluor 488 (trademark),hydrazide or 5-(or 6-)-carboxy-2', 7'-dichlorofluorescein may be mentioned. Among them, fluorescein is particularly preferred.
In the present invention, the concentration of the 1,2-dioxetane derivative to be used may appropriately be determined within a range where the measured value relative to standard solutions having a zero concentration and a positive concentrationof the object to be measured satisfies a measured value-to-concentration approximately linear relation. The concentration can suitably be set to a range of from 0.1 to 1.2 mM. In the concentration region having a remarkably lower concentration than theabove range, the measured value relative to the positive concentration tends to be on a plateau, and the upper limit of the measurement range tends to be low. On the contrary, in the concentration region having a remarkably higher concentration than theabove range, a background in the measured value relative to the zero concentration will increase, and the detection sensitivity in the low concentration region of the object to be measured tends to deteriorate.
The concentration of the cationic surfactant to be used is suitably within a range of from 0.0025% to 2.0%. In order to obtain the most suitable luminescence amount, it is particularly preferably from 0.01 to 0.10%. As for the concentration ofthe fluorescent material to be used, if the concentration of the 1,2-dioxetane derivative is high, it will be required to be proportionately high. However, it is preferably from about 0.0025 to about 0.25% in order to increase the sensitizing effect.
In the present invention, the cationic surfactant and the fluorescent material are made to coexist when the 1,2-dioxetane derivative of the formula 1 is made to generate chemiluminescence by means of the above activator. Accordingly, the same orlarger luminescence amount than that of a conventional method can be obtained.
The detection of the chemiluminescence is not particularly limited. For example, all the above respective components, namely, the 1,2-dioxetane derivative, the cationic surfactant, the fluorescent material and the activator are brought intocontact, followed by incubation, and the reaction solution is introduced into a luminescence detector after a certain period of time. As the luminescence detector, a luminometer provided with single photon counter is suitably used. When luminescencehaving high intensity is obtained by means of a sensitizer or the like, a silicon photodiode or a photographic film can be used. When an enzyme is used as the activator, an enzyme reaction stopper may be added before the reaction solution is introducedinto the luminescence detector.
Further, the chemiluminescence reaction which proceeds simultaneously with the progress of the enzyme reaction may be continuously monitored to measure the increasing rate of the luminescence amount. If the increasing rate is within a range of acertain period of time, it will not be required to strictly define the time between the beginning of the enzyme reaction i.e. the beginning of the chemiluminescence reaction and the luminescence detection. In a case where the chemiluminescence reactionis continuously monitored, the optimum pH of the enzyme reaction with a chemiluminescent substrate and the optimum pH of the chemiluminescence reaction which occurs successively preferably agree with each other as far as possible. For example, when theactivator is an alkaline phosphatase, the optimum pH of the enzyme reaction substantially agree with the optimum pH of the chemiluminescence reaction, and a 2-amino-2-methyl-1-propanol buffer solution or a diethanolamine buffer solution having a pH ofabout 10 may suitably be used.
On the other hand, the composition for chemiluminescence of the present invention is one comprising the 1,2-dioxetane derivative of the formula 1, the cationic surfactant and the fluorescent material. Further, it may appropriately contains a pHadjustor or a preservative such as sodium azide to secure the storage stability. The composition for chemiluminescence may be used as a solid, or it may be used in the form of a solution, for example, by dissolving it in an aqueous buffer solution.
The luminescence method according to the present invention makes it possible to obtain the same or larger luminescence amount than that of a conventional method. Particularly when tri-n-butylhexadecylphosphonium bromide is used as the cationicsurfactant, a luminescence amount larger than that of a conventional method can be obtained. Further, the composition for chemiluminescence according to the present invention, when the activator is made to act thereon, not only makes it possible toobtain the same or larger luminescence amount than that of a conventional method but also maintains extremely high storage stability.
Now, the present invention will be described in further detail with reference to Examples. However, it should be understood that the present invention is by no means limited to such specific Examples.
Examples 1 to 11
A 5-membered dioxetane phosphate (formal name: 5-t-butyl-4,4-dimethyl-l-(3'-phosphoryloxy)phenyl-2,6,7-trioxabicyclo[3.2- .0]heptane disodium salt) of the formula 3:
##STR00004## was produced in accordance with JP-A-9-216887 and purified by using a chelate resin. To a 0.1 to 0.2 M 2-amino-2-methyl-1-propanol buffer solution (pH 10.0) or diethanolamine buffer solution (pH 10.0) comprising 0.4 mM of thedioxetane phosphate thus obtained, fluorescein at a concentration as shown in Table 1 as a fluorescent material and 0.02% sodium azide, a cationic surfactant as shown in Table 1 was added to have a concentration as shown in Table 1. The solution isconsidered as a composition solution for chemiluminescence. Further, in Example 11, a commercially available Emerald-II™ (manufactured by TROPIX, INC.; containing poly(benzyltributyl)ammonium chloride as a cationic surfactant and sodium fluoresceinas a fluorescent material) was used as the fluorescent material and the cationic surfactant.
50 μL of the composition solution for chemiluminescence was reacted with 10 μL of a 50 mM Tris-HCl buffer solution containing 10-18 mol/mL of alkaline phosphatase, a 0.1% gelatin hydrolyzate, 1 mM MgCl2, 0.15 M NaCl and 0.05%sodium azide, and the luminescence amount was measured. Luminometer LB96V (detector: single photon counter; measured wavelength-region: from 380 to 630 nm) manufactured by BERTHOLD TECHNOLOGIES GmbH and Co, KG was used for the luminescence measurement,and as the luminescence measuring condition, the luminescence amount (count) was measured for 1 second 5 minutes after the composition for chemiluminescence and the above enzyme solution were mixed. The results are shown in Table 1. Further, the ratioof the luminescence amount based on the luminescence amount in Comparative Example 1 as mentioned hereinafter (in which the surfactant and the fluorescent material were not added) is shown as the amplification rate in the rightmost column in Table 1.
As evident from Table 1, the amplification rate of luminescence was at least 10 times in any case where the cationic surfactant was added. Further, when hexadecyltrimethylammonium bromide (CTAB) and tetradecyltrimethylammonium chloride which arequaternary ammonium salts having three methyl groups were compared with tetradecyldimethylbenzylammonium chloride (benzalkonium chloride) and hexadecyldimethylbenzylammonium chloride having one of the above methyl groups replaced by a benzyl group, thelatter showed higher amplification rates. That is, if the hydrophobicity of the substituents in the hydrophilic moiety in the vicinity of N.sup. is increased, the amplification rate of luminescence tended to increase.
Further, when triphenylphosphonium salts (ethyl, n-heptyl and n-tetradecyl) were compared with one another, such a result was shown that the longer the alkyl chain is, the higher the amplification rate of luminescence becomes. Whentri-n-butylhexadecylphosphonium bromide having a tri-n-butyl group having high hydrophobicity as the substituent in the hydrophilic moiety and having a hexadecyl group as a long chain alkyl group was used, the highest amplification rate of 430.49 timeswas recorded. This value was higher than the amplification rate of 97.53 times in a case where Emerald-II™ which is commercially available as the enhancer was used.
Comparative Examples 1 to 8
The same operation as in Example 1 was carried out except that the surfactant and the fluorescent material were not added in Comparative Example 1, and an anionic, nonionic or amphoteric surfactant as shown in Table 1 having a concentration asshown in Table 1 was used instead of the cationic surfactant in Comparative Examples 2 to 8. The results are shown in Table 1.
In the case where SDS (sodium dodecyl sulfate) as the anionic surfactant (Comparative Example 2); Triton X-100 (Comparative Example 3), Tween 80 (Comparative Example 4) or Tween 20 (Comparatives Example 5) as the nonionic surfactant; orn-dodecyldimethyl (3-sulfopropyl)ammonium hydroxide (Comparative Example 6), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) (Comparative Example 7) or CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l-propane sulfonate)(Comparative Example 8) as the amphoteric surfactant was used, the luminescence amplification rate was at most 5 times as compared with Comparative Example 1, in which the surfactant and the fluorescent material were not added, and substantially noeffect was obtained.
Comparative Examples 9 and 10
For comparison with commercial products, the luminescence amount was measured in each of cases where a chemiluminescence substrate CSPD (trademark) (manufactured by TROPIX, INC., a 1,2-dioxetane compound having no furan ring) was used instead ofthe composition solution for chemiluminescence of Example 1, and the surfactant and the fluorescent material were not used (Comparative Example 9), and CSPD (trademark) (manufactured by TROPIX, INC., a 1,2-dioxetane compound having no furan ring) havingEmerald II™ (manufactured by TROPIX, INC., containing poly(benzyltributyl)ammonium chloride as the cationic surfactant and sodium fluorescein as the fluorescent material) added thereto was used instead of the composition solution forchemiluminescence of Example 1 (Comparative Example 10). The results are shown in Table 2. The ratios of the luminescence amount of the former and the latter were shown in the rightmost column in Table 2 as the amplification rate.
TABLE-US-00001 TABLE 1 Fluorescent Surfactant material Lumines- Ampli- addition addition cence fication amount amount amount rate Surfactant (wt %) (wt %) (count) (times) Example 1 Benzethonium chloride 0.05 0.005 34,324 59.38 Example 2Hexadecyltrimethylammonium bromide 0.031 0.0025 90,913 157.29 (CTAB) Example 3 Tetradecyltrimethylammonium chloride 0.063 0.0025 91,496 158.30 Example 4 Tetradecyldimethylbenzylammonium 0.025 0.0025 102,067 176.59 chloride (benzalkonium chloride) Example5 Hexadecyldimethylbenzylammonium 0.025 0.0025 115,312 199.50 chloride (Cetol) Example 6 Tetra-n-butylphosphonium bromide 0.25 0.025 7,191 12.44 Example 7 Ethyltriphenylphosphonium bromide 1.25 0.025 6,960 12.04 Example 8 n-Heptyltriphenylphosphoniumbromide 1.25 0.025 12,395 21.44 Example 9 n-Tetradecyltriphenylphosphonium 0.025 0.0025 50,662 87.65 bromide Example 10 Tri-n-butylhexadecylphosphonium 0.025 0.0025 248,825 430.49 bromide Example 11 Poly(benzyltributyl)ammonium <0.2* <0.2* 56,37497.53 chloride Comparative Not added -- -- 578 -- Example 1 Comparative SDS (sodium dodecyl sulfate) 0.0025 0.025 1,463 2.53 Example 2 Comparative Triton X-100 0.0025 0.025 1,361 2.35 Example 3 Comparative Tween 80 0.0025 0.025 1,100 1.90 Example 4Comparative Tween 20 0.0025 0.025 1,125 1.95 Example 5 Comparative n-Dodecyldimethyl(3- 0.025 0.0025 988 1.71 Example 6 sulfopropyl)ammonium hydroxide Comparative CHAPS 0.92 0.0025 2,214 3.83 Example 7 Comparative CHAPSO 0.95 0.0025 2,464 4.26 Example 8*Calculated from catalog value
TABLE-US-00002 TABLE 2 Fluores- cent Types of Surfactant material Lumi- Amplifi- substrate/surfactant/ addition addition nescence cation fluorescent amount amount amount rate material (wt %) (wt %) (count) (times) Comparative CSPD (commercialproduct)/ -- -- 541 -- Example 9 Nil/Nil Comparative CSPD (commercial product)/ <0.2* <0.2* 164,088 303.30 Example 10 poly(benzyltributyl)ammonium chloride/sodium fluorescein *Calculated from catalog value
Examples 12 and 13
The composition solution for chemiluminescence prepared in Example 10 (Example 12) and the composition solution for chemiluminescence prepared in Example 4 (Example 13) were stored for 4 days under the light shielding condition in an incubator at4° C. and 40° C., respectively, for storage stability test. Then, 50 μL of each of the solutions was reacted with 10 μL of an enzyme solution containing 10-18 mol/mL of alkaline phosphatase, 50 mM of Tris-HCl, a 0.1% gelatinhydrolyzate, 1 mM MgCl2, 0.15 M NaCl and 0.05% NaN3, and the luminescence amount was measured. Luminometer LB96V manufactured by BERTHOLD TECHNOLOGIES GmbH and Co. KG was used for the luminescence measurement, and the luminescence amount(count) was measured for 1 second 5 minutes after the beginning of the above reaction as the luminescent measuring condition. The results are shown in Table 3. As evident from Table 3, the luminescence amounts were 97.5% (Example 12) and 101.5%(Example 13), respectively, even after storage for 4 days at 40° C., and substantially no decrease of the luminescence amount was observed.
Comparavetive Example 11
The same operation as in Example 12 was carried out except that CSPD (trademark) (manufactured by TROPIX) having Emerald-II™ added thereto, used in Comparative Example 10 was used instead of the composition solution for chemiluminescence. The results are shown in Table 3. As evident from Table 3, after storage for 4 days at 40° C., the luminescence amount deceased to 86.6% of that after storage for 4 days at 4° C.
TABLE-US-00003 TABLE 3 Comparative Example 12 Example 13 Example 11 Type of Substrate according Substrate according CSPD substrate to the present to the present invention* invention* Surfactant Tri-n- Tetradecyldimethyl- Poly(benzyl-butylhexadecyl benzylammonium tributyl) phosphonium chloride ammonium chloride bromide (benzalkonium chloride) Luminescence 4° C. 204,393 78,011 140,896 amount (1) (count) 40° C. 199,368 79,189 121,995 (2) (2)/(1) 97.5% 101.5% 86.6%*5-t-Butyl-4,4-dimethyl-1-(3'-phosphoryloxy)phenyl-2,6,7-trioxabicyclo[3.2- .0]heptane disodium salt
The entire disclosure of Japanese Patent Application No. 2003-304958 filed on Aug. 28, 2003 including specification, claims and summary are incorporated herein by reference in its entirety.
* * * * *
Field of SearchFluorescent label
With fluorescence or luminescence
OXIDATION - REDUCTION INDICATORS
One double bond between ring members of the hetero ring
Chalcogen attached indirectly to the hetero ring by nonionic bonding
Halogen attached directly or indirectly to the hetero ring by nonionic bonding
HIGH ENERGY SUBSTRATES (E.G., FLUORESCENT, CHEMILUMINESCENT, RADIOACTIVE, ETC.)